2 replies to this topic

[mhj222]

We have some genes that we want to test at higher levels of expression. Apart from knock-in, does anyone have any idea how to do this? SINEUP would be one way, but its quite a new technology so I was wondering if anyone has given it a try?

[Jon Moulton]

There is a new technique applicable for some genes -- inhibiting natural antisense transcripts to change regulation of their complementary mRNAs.

Inhibition of natural antisense transcripts in vivo results in gene-specific transcriptional upregulation.

Modarresi F, Faghihi MA, Lopez-Toledano MA, Fatemi RP, Magistri M, Brothers SP, van der Brug MP, Wahlestedt C.
Nat Biotechnol. 2012 Mar 25;30(5):453-9. doi: 10.1038/nbt.2158.
http://www.ncbi.nlm....pubmed/22446693

Another more established possibility, if the mRNA of interest is under miRNA repression, is to protect the site on the mRNA where the miRNA binds using an oligo as a target protector.

Target protectors reveal dampening and balancing of Nodal agonist and antagonist by miR-430.

Choi WY, Giraldez AJ, Schier AF.  Science. 2007 Oct 12;318(5848):271-4. Epub 2007 Aug 30.
http://www.ncbi.nlm....pubmed/17761850

[bob1]

Try an inducible promoter on an inserted plasmid.